The present invention, in some embodiments thereof, relates to isolated populations of renal progenitor cells and methods of generating and using same.
The fundamental functional unit of the kidney required for urine formation is the nephron. The number of nephrons in the human adult kidney (HAK) ranges from 300,000 to a million. The human metanephros (direct embryonic precursor tissue of the adult kidney) appears at the 5th week of gestation and renal stem/progenitor cells in a discrete region of the metanephros termed the metanephric/nephrogenic mesenchyme (MM) are induced to undergo mesenchymal-to-epithelial transition (MET) and form all epithelial cell types of the nephron until 34 weeks of gestation. These include glomerular (parietal, visceral) and tubular (proximal, distal) epithelia (which can be detected by segment-specific markers) but not collecting ducts. Thus, endowment of new nephrons is restricted to prenatal development in humans, while in rodents it persists only until the immediate postnatal period (up to 2 weeks postnatal).
Specifying the renal progenitor population throughout development are a unique combination of transcription factors, including the Hox11 paralogs, Osr1, Pax2, Eya1, Wt1, Sall1, Six2, and Cited1. These genes considered early markers of kidney progenitor cells are silenced, at least in part, by epigenetic modifications once nephrogenesis commences [1]. Epigenetic modifications responsible for transcriptional and lineage control are reversible and can be therefore manipulated. For example: methylation of lysine 27 on histone subunit H3 (H3K27me) by the Polycomb complex is associated with transcriptional repression, whereas methylation of lysine 4 on H3 (H3K4me) and any lysine acetylation of histones is associated with gene activation. Of all, recent experiments have established that Six2 and Osr1 are required to sustain a true committed stem cell in the MM that is capable of self-renewing and of differentiating towards different types of nephron epithelia [2-4].
Genetic diseases affecting the various cell types of the nephron are the leading cause of end-stage renal disease (ESRD) in the pediatric population which requires renal replacement therapy (dialysis, transplantation) and carries extremely high morbidity and mortality rates. Mutations in genes that specify the renal progenitor cell pool (see above) can lead to either congenital renal hypoplasia (too few nephrons) or to the appearance of malformed nephrons (congenital renal dysplasia). These can be viewed as ‘kidney stem cell diseases’. In contrast, mutations in genes that participate in the function of a highly specialized cell type in the kidney result in loss of that specific task. One such example are genetic defects in structural proteins of the glomerular podocyte (podocin, nephrin), an epithelial cell type responsible for the filtration barrier, which lead to massive spillage of protein to the urine and to the appearance of nephrotic syndrome, focal segmental glomerulosclerosis and ESRD (ref). In contrast to acquired childhood nephrotic syndrome which is sensitive to steroid treatment, the inherited disease is steroid-resistant.
The goal of renal regenerative medicine is to create an unlimited supply of human cells resembling the renal progenitors residing in the MM so as be able to replenish diseased ones in renal hypoplasia/dysplasia, affording enhanced nephron development or replace glomerular podocytes by inoculating a cell population containing the podocyte progenitor cell.
Derivation of renal stem/progenitors can be achieved by various strategies. One strategy is to sort them out from developing human kidneys via specific surface markers (similar to hematopoietic stem cells of the hematopoietic system). Another strategy is to induce differentiation of pluripotent cells (allogeneic human ES) along the renal lineage. A third strategy is to dedifferentiate autologous cell sources such as human adult kidney (HAK) cells into renal stem/progenitors.
Yamanaka and colleagues have shown the ability to reprogram cells into a pluripotent phenotype via the over-expression of specific factors, Oct4, c-Myc, Sox2, Klf4 [5]. Recently, Melton and colleagues reported the in vivo reprogramming of adult pancreatic exocrine cells to beta-cells using a strategy of re-expressing key developmental regulators of the endocrine pancreas [6], indicating a general paradigm for directing cell reprogramming and trans-differentiation across two mature cell types without reversion to a pluripotent stem cell state.
Metsuyanim et al teaches that the cessation of nephrogenesis is coincident with dramatic down-regulation of the renal progenitor genes, which is associated in part with epigenetic silencing [1].
U.S. Patent Application No. 20060177925 teaches multipotent renal progenitor cells (MRPC) that are antigen positive for vimentin and Oct-4, and are antigen negative for zona occludens, cytokeratin, and major histocompatibility Class I and II molecules.